Myelination allows for the conduction of rapid axonal conduction velocities without a concomitant increase in axon diameter. Myelin disorders such as multiple sclerosis represent the most common forms of neurological disorders in younger populations. Analysis of the pathologies associated with myelin disorders is complicated by the complex transcellular nature of myelination that largely restricts these analyses to animal models. Similarly, in vitro models have proven to be technically challenging, particularly for models of the CNS, and lack functional indicators for myelination or demyelination. In this application we propose to utilize new developments in surface chemistry, Bio-MEMs fabrication and neuroscience to engineer a hybrid system chip to carry out functional analysis of myelination and demyelination. The heart of this platform is a simple circuit between the motoneuron and myotubes cultured on silicon cantilevers, such that electrical stimulation of the motoneuron will cause contraction of the myotube with a corresponding deflection of the cantilever detectable by atomic force microscopy methodology. Photolithographic patterning will be used to guide the axons of the motoneurons through a myelination compartment, where Schwann cells (PNS) or oligodendrocytes (CNS) will be introduced to myelinate axonal segments. Functional read-outs of the consequences of myelination/demyelination will be provided not only by myotube contraction, but also by stimulating and recording microelectrodes flanking the myelination compartment, that allow for direct measurements of conduction velocity changes associated with myelination. Specific aims are as follows: 1) Fabricate and validate an in vitro hybrid model system for PNS myelination. In this aim we will: 1) Engineer a compartmentalized motoneuron-muscle circuit with a functional read-out for myotube contraction. 2) Introduce Schwann cells and characterize formation of PNS myelin. 3) Introduce stimulating MEAs and automated cantilever scanning into the system to allow for high efficiency monitoring of myelination associated functional changes. 2) Fabricate and validate an in vitro hybrid system model for CNS myelination. In this aim we will: 1) Determine myelination of a functional motoneuron-muscle circuit by oligodendrocytes. 2) Determine whether myelination by oligodendrocytes is enhanced in the presence of astrocytes. 3) Determine whether oligodendrocyte myelination is enhanced by activity in the motoneuron-muscle circuit. 3) Model functional affects of demyelination and remyelination using the in vitro hybrid system chip. In this aim we will: 1) Introduce a recording electrode into the hybrid system chip to record the first in vitro measurements of the effect of myelination on conduction velocity. 2) Correlate the functional consequences of demyelination with structural changes in the myelin sheath. 3) Use the hybrid system chip to monitor functional recovery associated with remyelination in vitro. We believe this myelination model system, once developed, will have a profound impact on researchers in the field of demyelinating disorders. PUBLIC HEALTH RELEVANCE: The proposed studies seek to develop a test platform for the functional analysis of myelination and demyelination. The results will provide a new foundation for understanding the pathologies associated with myelin disorders and for developing therapeutics aimed at alleviating these pathologies.